Comparative molecular and conventional cytogenetic analyses of three species of Rhinella (Anura; Bufonidae)

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella.


Introduction
The Bufonidae family is a monophyletic group of anurans, comprising 54 genera and 647 species, with native representatives distributed across almost every continent except in some countries, such as Australia, New Guinea, and Madagascar, where Rhinella marina was introduced [1,2].
In Brazil, Bufonidae is represented by eight genera, with Rhinella being the most representative, with 43 species distributed throughout the national territory [3].Owing to poorly clarified systematics and insufficient morphological, ecological, and molecular data, the genus Rhinella has undergone numerous taxonomic changes at both the interspecific and intraspecific levels.Discussions regarding the true taxonomic status of certain species have ensued, leading many to be classified within species complexes, thereby underscoring the taxonomic challenges associated with this genus [4][5][6][7][8].
Few studies have tackled the cytogenetics of the genus Rhinella, primarily involving conventional staining, banding, and few molecular analyses.These investigations revealed a remarkable conservatism in diploid numbers and chromosome morphology across the most distinct complexes, such as Rhinella marina, Rhinella margaritifera, Rhinella granulosa, and Rhinella crucifer [8,12,13].Notably, no divergences have been detected even in the patterns of C-banding, NOR (nucleolar organizer region), or fluorescence in situ hybridization (FISH) with probes from 18S rDNA [12,13].
An alternative approach to understanding the evolutionary mechanisms associated with karyotypic diversification is the analysis of different repetitive sequences, such as microsatellites, telomeric sequences, transposition elements, and more.These sequences play a crucial role in genome organization and plasticity and serve as excellent chromosomal markers in comparative cytogenomics [14][15][16][17][18].
Repetitive sequences are abundant in the genomes, and each species possesses a specific library of repetitive element families, categorized as satellite DNAs, minisatellites, microsatellites, transposable elements, and multigenic families of ribosomal RNA genes [19].Notably, microsatellite sequences have been highlighted for their significance.The physical mapping of the accumulation of microsatellite sequences has proven valuable in identifying sexual systems in amphibians, providing new insights into the mechanisms of genomic and karyotypic evolution [12,16,20].
In this study, we aimed to analyse the organization of repetitive DNA sequences in species representing the R. granulosa, R. margaritifera, and R. marina complexes using banding techniques and fluorescence in situ hybridization experiments, which contributed to a better understanding of karyotypic diversification and cytotaxonomy within the analysed species.
The specimens were euthanized with cutaneous applications of 2% lidocaine with the consent of the Ethical Committee in Animal Use (permission number 3539290620).Chromosome preparations were obtained from the intestinal epithelium and bone marrow following the protocols proposed by Ford and Hamerton [24] and Schmid [25], respectively.For male specimens, chromosomal preparations of gonads were also obtained from the testes according to Ford and Hamerton [24].For conventional cytogenetic analysis, chromosomes were stained with 5% Giemsa solution at pH 6.8 (0.5 ml of Giemsa supplemented with 10 ml of Phosphate buffer), while C-banding followed Sumner [26], with modifications in relation to the exposure time in barium hydroxide, where exposure varied between 1.5 minutes and 2 minutes, and the final staining where we used Wright stain.

Fluorescence in situ hybridization (FISH)
The 18S rDNA and telomeric sequences were amplified from the DNA of R. marina using the primers 18Sf (5'-CCGAGGACCTCACTAAACCA-3') and 18Sr (5'-CCGCTTTGGTGACTC TTGAT-3') [27], resulting in a 1400-bp PCR product.Telomeric (TTAGGG)n sequences were generated via PCR using the (TTAGGG) 5 and (CCCTAA) 5 primers without a DNA template, as described by Ijdo et al. [28].Because it is a highly conserved sequence among vertebrates, we opted not to sequence the 18S rDNA PCR product.The 18S rDNA and telomeric sequence probes were labelled by nick translation with digoxigenin-dUTP (Roche, Mannheim, Germany) following the manufacturer's recommendations.The signals from the probes were detected using an antidigoxin antibody with fluorescein (green) or rhodamine (red).FISH experiments with the aforementioned repetitive sequences were conducted following the protocol described by Yano et al. [29].

Microscopic analysis and image processing
A total of 20 metaphases per experiment were analysed to determine the diploid number, chromosome morphology, distribution of heterochromatic blocks, and patterns of distribution of the repetitive sequences.The metaphases with optimal dispersal were captured under a Leica 1000 DM microscope using a 100x objective.Karyotypes were organized using GenA-SIs software, version 7.2.6.19509(Applied Spectral Imaging, California, USA).The results of the FISH experiments were registered using a Zeiss Axio ImagerZ.2 epifluorescence microscope, and images were captured and edited with AxioVision 4.8 software (Zeiss, Jena, Germany).
Fundamental numbers (FNs) were calculated based on the total number of chromosome arms, considering metacentric (m), submetacentric (sm), and subtelocentric (st) as biarmed chromosomes and telocentric (t) as uniarmed chromosomes, according to the classification proposed by Green and Sessions [32].

FISH experiments
The 18S rDNA probe showed signals in the distal regions of the long arm of pair 5 of R. granulosa, in the interstitial region of the short arm of pair 7 in R. marina, and in the subdistal region of the short arm of pair 10 in R. margaritifera (

Discussion
The genus Rhinella comprises a great diversity organized into species complexes due to their high morphological similarity and complex phylogenetic relationships [4,11,[33][34][35].Although cytogenetic data obtained via classical approaches have been previously described for the analysed species, this study represents the first application of probes targeting different repetitive sequences to understand the genomic organization of Rhinella species.
The conserved karyotypic status observed among the species/complexes within the genus Rhinella has been a significant puzzle.Conventional chromosome analyses of the species in this study reaffirmed the conservation of the macrostructure of the Rhinella species karyotype.Bruschi et al. [13] reported a common karyotype with 2n = 22 and FN = 44 across all the species, albeit with minor variations in chromosome morphology.This observation led us to consider that the events resulting in morphological chromosomal changes occurred independently in each lineage of the species group, potentially involving the participation of repetitive sequences.
It is worth noting that the diploid number of 2n = 22 is also a recurrent finding in anurans in general, possibly corresponding to a plesiomorphic characteristic of the order [36].This chromosomal conservatism has evolutionary implications, as chromosomal characteristics can act as important pre-or postzygotic barriers to reproduction among distinct species [13,37].In this case, the chromosomal similarity between species would result in a relaxed isolation mechanism for speciation, contributing to the observed high frequency of hybridization events between species of the genus [11,13,33,34].
Moreover, other aspects of chromosome structure also exhibit uniformity in Rhinella.For example, although the PCR product has not been sequenced, our results with 18S rDNA corroborated previous data obtained from silver staining, confirming the presence of a nucleolar organizer region in pair 5 of R. granulosa, pair 7 of R. margaritifera, and pair 10 of R. marina [13].This allows inference of interspecific chromosomal homologies within species of this complex.This interspecific concordance is also observed in all species of the R. granulosa complex (distal portion of the long arm of pair 5), R. marina complex (interstitial portion of the short arm of pair 7), and R. margaritifera complex (subdistal portion of the short arm of pair 7 or 10), and suggests that the 18S rDNA probe obtained by PCR corresponded to the specific sequence of the genes [12,13,[38][39][40].
The dynamics of the location of 18S rDNA cluster probes across different species complexes may result from intra-and interchromosomal rearrangements, including inversions, fusions, and translocations, as well as transposition element-mediated transposition events or error reinsertion during amplification events [17,38,41,42].Therefore, this specificity within each group may represent a putative synapomorphy for each of them, except for the R. margaritifera group.In this group, NOR and 18S rDNA are found either in pair 7 or in pair 10, suggesting a reversion of the character or retention of the ancestral polymorphism, according to Bruschi et al. [13].
On the other hand, an alternative hypothesis that can be raised to justify these divergences in relation to the position of NORs and 18S rDNA is the variation in the copy number of tandem repeats/multigene families [43,44].Such variation can explain, for example, the differences observed both at the intraspecific level in R. margaritifera and at the interspecific level in Rhinella species, in which these markers are distributed at different positions.
Fornani et al. [44] reported that the differences in the number of copies of repetitive sequences of U1 and U2 snDNA were the result of the loss or reduction in the number of copies of these sequences in the different Xenopus (pipid frogs) species analysed.In the case of Rhinella species, the expansion of tandem repeats may have been an important driver of evolution following rearrangements such as translocation, inversion, deletion, and degeneration, which could explain the different locations of the repetitive sequences in the different Rhinella species.
Another informative chromosome marker in studies of karyotypic diversification in anurans is the distribution of heterochromatic blocks.Heterochromatin can serve as a hotspot for chromosomal rearrangements, and therefore, a detailed analysis of its composition and distribution enhances our understanding of karyotype evolution dynamics [12,18,20,45].Although C-banding analyses in species of the genus Rhinella are relatively limited, studies up to the level of the family Bufonidae suggest a highly conserved banding pattern, with these blocks primarily restricted to centromeres and pericentromeric regions [12,46].
While accumulations of heterochromatin in pericentromeric regions in pairs 3 and 6 in R. marina and pairs 4 and 6 in R. margaritifera may suggest rearrangements, studies with species of the genus Rhinella and other Bufonids have considered such findings as potential population markers within Bufonidae [47,48].Notably, extensive heterochromatic blocks observed in the chromosome pairs of the species R. marina and R. margaritifera indicate the amplification of repeat units, underscoring the role of repetitive DNAs in Rhinella chromosome evolution and, consequently, in karyotypic divergences among species [49].
In recent years, several studies have reported that certain species exhibit specific markers that may play regulatory roles in gene activities and genomic functions [50,51].In the case of species of the genus Rhinella, despite is phylogenetically related, and diverse patterns in the location of microsatellite repeats have been identified.These differences suggest potential variations in evolutionary events of genomic organization, with some microsatellite accumulations being species-specific (Fig 8) [52,53].
Studies have reported that microsatellites are not randomly distributed in eukaryotic genomes and may be in the same chromosomal locations in closely related species [18,54,55].Indeed, the distributions of microsatellites in the species R. marina and R. granulosa, which are phylogenetically more closely related, were more similar than those in R. margaritifera, which occupies a more basal position in the phylogeny of the genus Rhinella, displaying more distinct patterns of microsatellite distribution.These results reinforce the hypothesis that microsatellite distribution can provide phylogenetic markers depending on the groups and species studied [11].
The specific accumulation of microsatellites on heteromorphic sex chromosomes is common due to the appearance of nonrecombinant regions.In addition, significant accumulations of microsatellite sequences can also occur in euchromatic regions and not necessarily in sexlinked regions/chromosomes, and in turn, such cytogenetic markers could play a role in modulating genomic function [17,51,56].Given this context, two interesting aspects should be raised: 1-the accumulation of microsatellite sequences in pair 1 of R. marina and R. margaritifera; 2-the dimorphism of pair 10 of R. margaritifera, as well as the accumulation of microsatellites in this same pair in both R. granulosa and R. margaritifera.
Specific accumulations in pair 1 have been reported in some species of Bufonidae [57,58].Interestingly, molecular studies have revealed that in four species of the genus Bufo, genes associated with sex definition are present on chromosome pair 1 [57,59].Furthermore, a recent study based on genomic data revealed numerous sex-linked markers, located throughout chromosome 1, with some markers also linked to chromosome 7. Overall, this provides strong support for a genetic sex determination system on chromosome 1 [58].However, no information on the accumulation of repetitive sequences or sex-defining genes in pair 10 of Bufonidae has been described.Unfortunately, the lack of genomic data available for the species analysed limits us from suggesting that such markers may have some functionality in identifying sex chromosomes in Rhinella species and that more sophisticated genomic analyses, such as comparative genomic hybridization or next-generation sequencing, should be carried out to address these uncertainties.
Interestingly, in the karyotypes of the three species, the trinucleotide probes (CAC) 10 , (CAT) 10 , and (GAG) 10 showed a dispersed distribution pattern throughout the chromosomes.Such a distribution of microsatellite sequences throughout genomes has been associated with the activity of transposable elements, which may contain microsatellite repeats in their sequences, thus contributing to the dispersion of units during transposition events and influencing the karyotypic diversification processes of the species [60,61].
In summary, our data suggest that repetitive DNAs play a dynamic role in chromosomal changes in Rhinella, influencing the chromosomal microstructure and contributing to our understanding of the evolutionary mechanisms that led to karyotypical diversification in distinct phylogenetic groups within this genus.

Fig 1 .
Fig 1. Specimen collection sites of R. granulosa, R. margaritifera, and R. marina in the Amazon rainforest, Para ´, Brazil.The red triangles highlight the collection sites of the species analysed in this study.Map produced in QGIS software, version 3.36 (https://qgis.org/pt_BR/site/),input data are public domain obtained from the Instituto Brasileiro de Geografia e Estatı ´stica (https://www.ibge.gov.br/geociencias/downloads-geociencias.html).https://doi.org/10.1371/journal.pone.0308785.g001 Fig 3).Hybridization with telomeric sequence probes produced signals exclusively in the distal region of the chromosomes (Fig 4).The microsatellite probes produced two different patterns of hybridization in Rhinella species: scattered signals or signals in specific regions of the chromosome.In R. granulosa, nine probes produced signals.In general, all the probes hybridized to the distal portion of all the chromosomes, with some probes also showing chromosome-specific signals (Fig 5).The

Fig 5 .
Fig 5. Distribution of microsatellites in the genome of R. granulosa.The microsatellite probes used are indicated at the top left.The arrows indicate the chromosomes that showed specific signs of hybridization with the microsatellite probe used.The chromosomes were counterstained with DAPI (blue).Scale bar = 10 μm.https://doi.org/10.1371/journal.pone.0308785.g005

Fig 6 .
Fig 6.Distribution of microsatellites in the genome of R. margaritifera.The microsatellite probes used are indicated at the top left.The arrows indicate the chromosomes that showed specific signs of hybridization with the microsatellite probe used.The chromosomes were counterstained with DAPI (blue).Scale bar = 10 μm.https://doi.org/10.1371/journal.pone.0308785.g006

Fig 7 .
Fig 7. Distribution of microsatellites in the genome of R. marina.The microsatellite probes used are indicated at the top left.The arrows indicate the chromosomes that showed specific signs of hybridization with the microsatellite probe used.The chromosomes were counterstained with DAPI (blue).Scale bar = 10 μm.https://doi.org/10.1371/journal.pone.0308785.g007

Fig 8 .
Fig 8. Distribution scheme of the main microsatellites on the autosomal chromosomes of a) R. granulosa, b) R. margaritifera, and c) R. marina.The red markers indicate the distribution of the microsatellites specific to each chromosome.The black markers indicate the centromeric position.https://doi.org/10.1371/journal.pone.0308785.g008